Inhibition of human teomerase by a G-quadruplex-interaction compound

ABSTRACT

Certain non-nucleoside compounds that will selectively inhibit telomerase by targeting the nucleic add structures, such as G-quadruplexes, that may be associated with human telomeres or telomerase have been identified. Inhibition of human telomerase by two perylenetetracarboxylic acid diimides and a carbocyanine has been demonstrated.  1 H-NMR studies have evidenced the stabilization of a G-quadruplex by the perylenetetracarboxylic acid diimide compounds and provided evidence that these and structurally related compounds inhibit the telomerase enzyme by a mechanism consistent with interaction with G-quadruplex structures.

[0001] The present application claims the priority of co-pending U.S.Provisional Patent Application Ser. No. 60/073,629, filed Feb. 4, 1998,the entire disclosure of which is incorporated herein by referencewithout disclaimer. The government may own rights in the presentinvention pursuant to contract number U19CA-67760-O₂, and contractnumber NCDDG, CA67760 from the National Cancer Institute, and contractnumber CA49751 and contract number CA77000 from the National Institutesof Health.

BACKGROUND OF THE INVENTION

[0002] I. Field of the Invention

[0003] This invention relates to the field of cancer therapy. Theinvention also relates to screening methods for identifyingpharmacologically active compounds that may be useful for treatingproliferative diseases. More particularly, the inventors have identifiednon-nucleoside molecule compounds that interact with specific DNAstructures and which inhibit human telomerase.

[0004] II. Description of Related Art

[0005] Cancer, which is a cell proliferative disorder, is one of theleading causes of disease, being responsible for 526,000 deaths in theUnited States each year (Boring et al., 1993). For example, breastcancer is the most common form of malignant disease among women inWestern countries and, in the United States, is the most common cause ofdeath among women between 40 and 55 years of age (Forrest, 1990). Theincidence of breast cancer is increasing, especially in older women, butthe cause of this increase is unknown. Malignant melanoma is anotherform of cancer whose incidence is increasing at a frightening rate, atleast sixfold in the United States since 1945, and is the single mostdeadly of all skin diseases (Fitzpatrick, 1986).

[0006] One of the devastating aspects of cancer is the propensity ofcells from malignant neoplasms which disseminate from their primary siteto distant organs and develop into metastatic cancers. Animal testsindicate that about 0.01% of circulating cancer cells from solid tumorsestablish successful metastatic colonies (Fidler, 1993). Despiteadvances in surgical treatment of primary neoplasms and aggressivetherapies, most cancer patients die as a result of metastatic disease.Hence, there is a need for new and more efficacious cures for cancer.

[0007] The ends of chromosomes have specialized sequences, termedtelomeres, comprising tandem repeats of simple DNA sequences. Humantelomeres consist of the sequence 5′-TTAGGG (Blackburn, 1991; Blackburnet al., 1995). Telomeres have several functions apart from protectingthe ends of chromosomes, the most important of which appear to beassociated with senescence, replication, and the cell cycle clock(Counter et al., 1992). Progressive rounds of cell division result in ashortening of the telomeres by some 50-200 nucleotides per round. Almostall tumor cells have shortened telomeres, which are maintained at aconstant length (Allshire et al., 1988; Harley et al., 1990; Harley etal., 1994) and are associated with chromosome instability and cellimmortalization.

[0008] The enzyme telomerase adds the telomeric repeat sequences ontotelomere ends, ensuring the net maintenance of telomere length in tumorcells commensurate with successive rounds of cell division. Telomeraseis a DNA polymerase with an endogenous RNA template (Feng et al., 1995),on which the nascent telomeric repeats are synthesized. A significantrecent finding has been that approximately 85-90% of all human cancersare positive for telomerase, both in cultured tumor cells and primarytumor tissue, whereas most somatic cells appear to lack detectablelevels of telomerase (Kim et al., 1994; Hiyama et al., 1995a). Thisfinding has been extended to a wide range of human tumors (see, forexample, references Broccoli, 1994 and Hiyama et al., 1995b) and islikely to be of use in diagnosis.

[0009] Human telomerase has been proposed as a novel and potentiallyhighly selective target for antitumor drug design (Feng et al., 1995;Rhyu et al., 1995; Parkinson, 1996). Studies with antisense constructsagainst telomerase RNA in HeLa cells show that telomere shortening isproduced, together with the death of these otherwise immortal cells(Feng et al., 1995). Sequence-specific peptide-nucleic acids directedagainst telomerase RNA have also been found to exert an inhibitoryeffect on the enzyme (Norton et al., 1996).

[0010] Among chemical agents, 2,6-diamido-anthraquinones have beenreported as DNA-interactive agents (Collier and Neidle, 1988; 1992;Agbandje et al., 1992). These compounds have been shown to act asselective DNA triplex interactive compounds (Fox et al., 1995; Haq etal., 1996), with reduced affinity for duplex DNA and only moderateconventional cytotoxicity in a range of tumor cell lines. A carbocyaninedye, 3,3′-diethyloxadicarbocyanine (DODC,), has been reported to binddimeric hairpin G-quadruplex structures (Chen et al., 1996).

[0011] This invention describes a novel class of non-nucleosidemolecules that are telomerase inhibitors. These compounds havedemonstrated their ability to interact with telomeres which formstructures called the G-quadruplex structures. As telomeres are involvedin controlling the cell cycle, cell replication and aging, theseinhibitors of telomerase prevent uncontrolled cell growth and theimmortality of tumor cells.

SUMMARY OF THE INVENTION

[0012] The present invention has demonstrated for the first time that anon-nucleoside, small molecule can target the G-quadruplex structure andcan act as a telomerase inhibitor. Accordingly, methods have beendeveloped that identify these classes of compounds and severalinhibitors identified.

[0013] Compounds such as those described here, which interactselectively with G-quadruplex structures and inhibit telomerase, areexpected to be useful as inhibitors of the proliferation of cells thatrequire telomerase to maintain telomere length for continued growth. Theinvention thus relates to novel methods for identifying compounds thatwill be useful in this regard, and also includes new classes oftelomerase inhibitors. In this regard, several perylene compounds andcarbocyanines have been shown to interact with G-quadruplex structures.Since telomerase appears to be found almost exclusively in tumor cells,these agents are contemplated to be useful as antitumor agents.

[0014] In one aspect of the invention, compounds that act as telomeraseinhibitors have been identified. It has been found that compounds thatbind to the human G-quadruplex structure inhibit the human telomerase.The identification of such G-quadruplex interactive agents is anefficient approach for identifying human telomerase inhibitors. Methodsfor identifying these G-quadruplex interactive agents includeidentifying compounds whose three-dimensional structure is complementaryto that of the G-quadruplex structure. Another method for identifyingG-quadruplex interactive compounds is to identify compounds thatinteract with G-quadruplexes using such methods as dye displacement ormelting points of G-quadruplex/compound hybrids.

[0015] More particularly, candidate compounds that inhibit telomeraseactivity are identified by first obtaining the three-dimensionalstructure of a compound that might interact with theG-quadruplex.selected compound. The complementarity of the compound tohuman telomere DNA G-quadruplex is then determined. If there is a highdegree of complementarity, telomerase inhibition activity is indicated.

[0016] Alternatively, one can contact a telomerase inhibitor candidatecompound with human DNA G-quadruplex; and then determine the meltingpoint of the human DNA G-quadruplex. The inventors have found that anincrease in melting point of the quadruplex indicates telomeraseinhibitory activity of the compound.

[0017] Additionally, telomerase inhibitors may be identified by firstpreparing a DNA G-quadruplex/dye complex with a dye intercalated intothe G-quadruplex; then contacting complex with a telomerase inhibitorcandidate. Displacement of the dye in the complex identifies thecandidate as a telomerase inhibitor.

[0018] Yet another aspect of this invention is to provide non-nucleosideinhibitors of telomerase. Using the disclosed screening methods,compounds have been identified that bind to human G-quadruplexstructures. The invention includes perylene compounds, exemplified byN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide that are useful telomerase inhibitors. Novel compounds such asN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimideare also within the scope of the invention.

[0019] A preferred G-quadruplex structure is formed from the sequenced(AGGGTTAGGGTTAGGGTTAGGG) or the sequences d(TTAGGG)₄, d(TAAGGGT)₄, ord(TTAGGGTT)₄ either alone or in the presence of a G-quadruplexinteractive perylene diimide of general structure I. The structures weredetermined by NMR spectroscopy. Alternatively, one may determine thethree-dimensional structure of potential G-quadruplex interactive agentsby x-ray diffraction or molecular mechanics calculations. Preferredprograms for determining the degree of complementarity between thepotential G-quadruplex interactive agent and these G-quadruplexstructures include DOCK, autoDOCK, AMBER and SYBYL. The preferredmethods for generating orientations between the potential G-quadruplexinteractive agents and these G-quadruplex structures are manual andusing the DOCK or autoDOCK programs. The cutoff for determining thelikelihood that the orientation of the potential G-quadruplexinteractive agent and the G-quadruplex structure have sufficientchemical interaction to form a complex is roughly 75% of the favorableintermolecular interaction energy, calculated with the above programs,of the perylene diimide 2-d(TTAGGG)₄ complex structure as determined byNMR spectroscopy.

[0020] Preferred G-quadruplex structures are those formed by thesequences d(TTAGGG)₄, d(AATGGGT)₄ and d(TTAGGGTT)₄. Several methods ofdetermining the interaction of potential G-quadruplex interactive agentswith these structures include UV/VIS spectroscopy, in which the changesin the UV/VIS spectrum of the potential agent under more than a 10%change at the wavelength due solely to the ligand and which undergoesthe most change, upon addition of an excess of the G-quadruplexstructure; UV spectroscopy, in which the melting temperature of theG-quadruplex structure as determined by a hyperchromicity transition ata given temperature range is increased by >5° C. upon addition of anexcess of the agent; UV/VIS spectroscopy in which addition of apotential G-quadruplex interactive agent to a complex of aG-quadruplex-interactive perylene diimide and a G-quadruplex producesa >25% change in the absorption of due to the G-quadruplex-interactiveperylene diimine-G-quadruplex complex; UV/VIS spectroscopy in whichaddition of a potential G-quadruplex interactive agent to a complex of aG-quadruplex-interactive carbocyanine and a G-quadruplex produces a >25%change in the absorption of due to the G-quadruplex-interactivecarbocyanine-G-quadruplex complex; NMR spectroscopy in which the meltingtemperature of the G-quadruplex as determined by the disappearance ofthe imino proton signals of the G-quadruplex is increase by >5° C. inthe presence of one- to two-equivalents of the agent; NMR spectroscopyin which the interaction of the agent with the G-quadruplex structure isdetermined by the shift of at least one of the imino protons of theG-quadruplex by >0.01 ppm upon addition of one- to two-equivalents ofthe agent; fluorescence spectroscopy in which the fluorescence emissionspectrum of the agent undergoes a shift of >5 nm and/or a change inintensity of >25% upon the addition of an excess of the G-quadruplexstructure; fluorescence spectroscopy in which the fluorescence emissionspectrum of a G-quadruplex-interactive perylene diimide-G-quadruplexcomplex undergoes a >25% change upon the addition of an excess of theagent; or fluorescence spectroscopy in which the fluorescence emissionspectrum of a G-quadruplex-interactive carbocyanine-G-quadruplex complexundergoes a >25% change upon the addition of an excess of the agent.

[0021] The preferred embodiments of the invention as it related to oneclass of G-quadruplex interactive telomerase inhibitors are compounds ofthe structure I in which

[0022] in which R¹ and R⁴ are independently taken from the set ofsub-structures given by the formula -L-A in which L is a linking grouptaken from the set consisting of:

[0023] where n is 1, 2, or 3; and each R5 is independently taken fromthe set H, Me, OH, or OMe;

[0024] where R5 is as before and Y is taken from the set O, S, SO, SO2,NH, NMe, NCOMe;

[0025] where R5 and Y are as before and X is taken from the set CH2, O,S, SO, SO2, NH, NMe, NCOMe;

[0026] where R6, R7, R8, and R9 are independently taken from the setconsisting of H, OMe, OEt, halo, or Me;

[0027] or a bond;

[0028] and A is taken from the set consisting of:

[0029] where m is 0-5 and each R6 is taken from the set consisting ofhalo, NH2, NO2, CN, OMe, SO2NH2, amidino, guanidino, or Me;

[0030] where o is 0 or 1; p is 0, 1, or 2; q is 1 or 2such that o+q iseither 2, in which case a pyrrolidine or pyrrole ring is indicated, or3, in which a piperidine or pyridine ring is indicated; r is 0, 1, 2, or3; R7 is H or Me; each R8 is independently taken from the set consistingof Me, NO2, OH, CH2OH, halo, or when r is 2 or 3, two adjacent R8substituents may be together taken as —(CH═CH)2- or —(CH2)4- such as toform an annulated six-membered ring;

[0031] where each R9 is independently taken from the set consisting ofH, Me, or both R9 can taken together be ═O; s is equal to 0 or 1; and Zis taken from the set consisting of CH2, 0, NH, NMe, NEt, N(Me)₂,N(Et)2, or NCO2Et;

[0032] where Q is either N, CH, NMe, or NEt; X is either O, S, NH, NMeor NEt; R10 and R11 are independently taken from the set consisting ofH, Me, CH2CO2Et, or R10 and R11 taken together consist of —(CH═CH)2- or—(CH2)4-;

[0033] where t is equal to 1, 2, 3, or 4; u is equal to 0, 1, 2, 3, or4, and each R12 is individually taken from the set consisting of Me, orOH;

[0034] OH, CO2R¹³, CON(R¹³)_(2,) SO3H, SO2N(R¹³)₂, CN, CH(CO2R¹³)₂,CH(CON(R¹³)₂)₂, N(R¹³)₂, or N(R¹³)₃ where R13 is either H, Me, Et, orCH2CH₂OH;

[0035] R2, R2′, R2″, R2″; R3, R3′, R3″, R3′″ are each independentlytaken from the set H, OMe, halo, or NO2.

[0036] In addition, this invention includes the development of otherG-quadruplex interactive telomerase inhibitors compounds derived fromstructure I, having the following structures:

[0037] The preferred embodiment of the invention as it relates toanother class of G-quadruplex interactive telomerase inhibitors arecompounds of the general structure II:

[0038] in which C is either a bond, —CH═CH—, —(CH═CH)2-, —(CH═CH)3-,p-phenylene, o-phenylene, p-phenylene-CH═CH—, or o-phenylene-CH═CH—; Bis O, S, or NR, and R is either Me or Et.

[0039] In addition, this invention includes the development of anotherG-quadruplex interactive telomerase inhibitor compound derived fromstructure II, having the following structure:

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0041]FIG. 1. Effect of increasing concentrations ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide on inhibition of telomerase catalyzed extension of an 18-merprimer d[TTAGGG]₃ (1 μM). Elongated primer was labeled with 1.5 μM of[α-³²P]-dGTP (800 Ci mmol⁻¹, 10 mCi ml⁻¹) with 1 mM DATP and dTTP usinga standard telomerase assay. Lanes 1-5 are 0, 10, 50, and 100 μM ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide.

[0042]FIG. 2. Changes in the UV/VIS absorbance spectrum ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide upon addition of increasing amount of [d(TTAGGGT)]₄, anoligodeoxyribonucleotide which adopts a G-quadruplex structure.

[0043]FIG. 3. Titration of [d(TTAGGG)]₄ withN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide. Imino proton region of the 500-MHz ¹H NMR is shown withincreasing amounts of added ligand. The resonances labeled G4*, G5*, andG6* represent resonances of final 2:1 ligand/G-quadruplex complexes.

[0044]FIG. 4. NMR-based model of[d(TTAGGG)]₄—N,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylicacid diimide complex. The ligand is stacked under the G6 guanine tetradwith positively charged side chains located in the grooves.

[0045]FIG. 5. Shows the design of an intramolecular quadruplex DNA thatcontains the human telomere repeats.

[0046]FIG. 6. Photocleavage of G4A DNA by TMPyP⁴ in K⁺ buffer.

[0047]FIG. 7. Model depicting G-quadruplex structure blocking primerextension by DNA polymerase.

[0048]FIG. 8. Primer extension of PQ sequence in the presence ofcompounds. Lane 1: water control; Lane 2: 50 mM K+; Lanes 3-6; QQ23;Lanes 7-10; QQ30; Lanes 11-14; QQ31; Lanes 15-18; APPER(Bis[1-(2-aminoethyl)piperdine]-3,4,9,10-perylenetetracarboxylicdiimide); Lanes 3,7,11, and 15; 0.5 μM; Lanes 4,6,12, and 16; 1 μM;Lanes 5,9,13 and 17; 10 μM; Lanes 6,10, 14 and 18; 50 μM.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0049] I. The Present Invention

[0050] A structure-based approach to discovering non-nucleosidecompounds that will selectively inhibit human telomerase by targetingthe nucleic acid structures, such as G-quadruplexes, that may beassociated with human telomeres or telomerase has been utilized.Inhibition of human telomerase by a 2,6-diamido anthraquinone has beensuccessfully demonstrated. ¹H-NMR has demonstrated the stabilization ofa G-quadruplex by this compound and evidence has been provided that thiscompound inhibits the telomerase enzyme by a mechanism consistent withinteraction with G-quadruplex structures. The present work shows thatnon-nucleoside, small molecules can interact with G-quadruplexes andinhibit telomerase.

[0051] Using the methods described, it was found that compounds thatbind to the human G-quadruplex structure inhibit the human telomerase.The identification of such G-quadruplex interactive agents is a noveland efficient approach for identifying human telomerase inhibitors.

[0052] It is envisioned that the telomerase inhibitors will providetherapy for tumors and cancers including skin cancers, connective tissuecancers, adipose cancers, breast cancers, lung cancers, stomach cancers,pancreatic cancers, ovarian cancers, cervical cancers, uterine cancers,anogenital cancers, kidney cancers, bladder cancers, colon cancers,prostate cancers, central nervous system (CNS) cancers, retinal cancer,blood, lymphoid cancers and the like.

[0053] II. Telomerase

[0054] Telomerase is a ribonucleoprotein enzyme that synthesizes onestrand of the telomeric DNA using as a template a sequence containedwithin the RNA component of the enzyme. The ends of chromosomes havespecialized sequences, termed telomeres, comprising tandem repeats ofsimple DNA sequences which in humans is 5′-TTAGGG (Blackburn, 1991;Blackburn et al., 1995). Apart from protecting ends of chromosomestelomeres have several other functions, the most important of whichappear to be associated with replication, regulating the cell cycleclock and ageing (Counter et al., 1992). Progressive rounds of celldivision shorten telomeres by 50-200 nucleotides per round. Almost alltumor cells have shortened telomeres, which are maintained at a constantlength (Allshire et al., 1988; Harley et al., 1990; Harley et al., 1994)and are associated with chromosome instability and cell immortalization.

[0055] With regard to human cells and tissues telomerase activity hasbeen identified in immortal cell lines and in ovarian carcinoma but hasnot been detected at biologically significant levels (that are requiredto maintain telomere length over many cell divisions) in mortal cellstrains or in normal non-germline tissues (Counter et al., 1992; Counteret al, 1994). These observations suggest telomerase activity is directlyinvolved in telomere maintenance, linking this enzyme to cellimmortality.

[0056] As described above, the immortalization of cells involves theactivation of telomerase. More specifically, the connection betweentelomerase activity and the ability of many tumor cell lines, includingskin, connective tissue, adipose, breast, lung, stomach, pancreas,ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervoussystem (CNS), retina and blood tumor cell lines, to remain immortal hasbeen demonstrated by analysis of telomerase activity (Kim, et al.,1994). This analysis, supplemented by data that indicates that theshortening of telomere length can provide the signal for replicativesenescence in normal cells, see PCT Application No. 93/23572,incorporated herein by reference, demonstrates that inhibition oftelomerase activity can be an effective anti-cancer therapy. Thus,telomerase activity can prevent the onset of otherwise normalreplicative senescence by preventing the normal reduction of telomerelength and the concurrent cessation of cell replication that occurs innormal somatic cells after many cell divisions. In cancer cells, wherethe malignant phenotype is due to loss of cell cycle or growth controlsor other genetic damage, an absence of telomerase activity permits theloss of telomeric DNA during cell division, resulting in chromosomalrearrangements and aberrations that lead ultimately to cell death.However, in cancer cells having telomerase activity, telomeric DNA isnot lost during cell division, thereby allowing the cancer cells tobecome immortal, leading to a terminal prognosis for the patient.

[0057] Methods for detecting telomerase activity, as well as foridentifying compounds that regulate or affect telomerase activity,together with methods for therapy and diagnosis of cellular senescenceand immortalization by controlling telomere length and telomeraseactivity, have also been described elsewhere.

[0058] A. G-Quadruplex Structures

[0059] Human telomeres form structures known as G-quadruplexes. Humantelomeres contain numerous repeats of the sequence TTAGGG, exhibiting anenhancement of G and T residues and a paucity of A residues.Intramolecular G-quadruplex DNA may be designed by generating a sequenceof human telomere repeats (FIG. 5). The G tetrad consists of four Gbases hydrogen bonded in Hoogsteen fashion symmetrically disposed abouta central axis, as shown in FIG. 5.

[0060] G-rich DNA is known to assume highly stable structures formed byHoogsteen base pairs between guanine residues (Williamson, 1994; Nadelet al., 1995). These structures, known as G-quadruplexes, are stabilizedin the presence of K⁺ and may have biological roles that are yet to bedetermined (Henderson et al., 1987; Hardin et al., 1997; Williamson etal., 1989). One particular region of the genome where these structuresmay play a significant biological role is at the ends of chromosomeswhere G-rich DNA is normally found (e.g., TTAGGG and TTGGGG tandemrepeats in human cells and ciliate Tetrahymena, respectively) (Hendersonet al., 1987; Blackburn and Greider, 1995; Sundquist and Heaphy, 1993).In addition, a number of genes containing G-rich DNA have beenidentified recently, and it has been proposed that the G-rich regionswithin these genes may regulate gene expression by forming G-quadruplexstructures (Sen and Gilbert, 1988; Hommond-Kosack et al., 1993; Murchieand Lilley, 1992; Simonsson et al., 1998). One potential biologicallyrelevant role of G-quadruplex DNA is as a barrier to DNA synthesis(Howell et al., 1996). This barrier has been thoroughly investigated andhas been found to be K⁺ dependent (Woodword et al., 1994). Thisobservation strongly suggests that the formation of G-quadruplex speciesis responsible for the observed effect on DNA synthesis (Weitzmann etal., 1996).

[0061] The inventors have shown that the 2,6-iamidoanthraquinoneBSU-1051 modulates human telomerase activity by a mechanism that isdependent on the elongation of the telomeric primer d(TTAGGG)₃ to alength that is then capable of forming an intramolecular G-quadruplexstructure (Sun et al., 1997). The inventors have also shown thatBSU-1051, by virtue of its interaction with G-quadruplex DNA, enhancesthe block of DNA synthesis by the G-quadruplex structure in the presenceof K⁺.

[0062] B. Methods for Identifying G-quadruplex Interactive Agents

[0063] Several methods for identifying classes of G-quadruplexinteractive agents may be employed. One method involves identifyingcompounds whose three-dimensional structure is complementary to that ofthe G-quadruplex structure. G-quadruplex structure is understood to meanat least in one sense the structure of the G-quadruplex that is formedby the single-stranded DNA corresponding to at least four repeats of thetelomeric sequence. In humans, the telomeric sequence is d(TTAGGG).Thus, the G-quadruplex structure of interest for the identification ofhuman telomerase inhibitors may be any sequence of the form{d([N₁]TAGGG[N₂])}₄ where [N₁] is zero to two bases corresponding to thehuman telomeric sequence; for example, [N₁] may equal G, GG, or may beabsent; where [N₂] is zero to three bases corresponding to the humantelomeric sequence; for example, [N₂] can equal T, TT, TTA or it may beabsent.

[0064] Alternatively, G-quadruplex structure is understood to mean thefold-over or intramolecular G-quadruplex formed from at least fourrepeats of the G-triad of telomeric sequence. Thus, the G-quadruplexstructure of interest for the identification of human telomeraseinhibitors may be any sequence of the form d([N₃][TTAGGG]₃[N₂]) where[N₂] is as defined above and [N3] is three G's preceded by zero to threenucleotides corresponding to the human telomeric sequence. Thesestructures may be determined by a variety of techniques includingmolecular mechanics calculations, molecular dynamics calculations,constrained molecular dynamics calculations in which the constraints aredetermined by NMR spectroscopy, distance geometry in which the distancematrix is partially determined by NMR spectroscopy, x-ray diffraction,or neutron diffraction techniques. In the case of all these techniques,the structure can be determined in the presence or absence of anyligands known to interact with G-quadruplex structures, including butnot limited to potassium and other metal ions,2,6-diamidoanthraquinones, perylene diimides, or carbocyanines.

[0065] Complementary is understood to mean the existence of a chemicalattraction between the G-quadruplex interactive agent and theG-quadruplex. The chemical interaction may be due to one or a variety offavorable interactions, including ionic, ion-dipole, dipole-dipole, vander Waals, charge-transfer, and hydrophobic interactions. Each of thesetype of interactions, alone or together, may be determined by existingcomputer programs using as inputs the structure of the compound, thestructure of the G-quadruplex, and the relative orientation of the two.Such computer programs include but are not limited to AMBER, CHARMM,MM2, SYBYL, CHEMX, MACROMODEL, GRID, and BioSym. Such programs arecontemplated as being useful for the determination of the chemicalinteraction between two molecules, either isolated, or surrounded bysolvent molecules, such as water molecules, or using calculationaltechniques that approximate the effect of solvating the interactingmolecules. The relative orientation of the two can be determinedmanually, by visual inspection, or by using other computer programswhich generate a large number of possible orientations.

[0066] Examples of computer programs include but are not limited to DOCKand AutoDOCK. Each orientation can be tested for its degree ofcomplementarity using the computer programs. An advantage of this methodis that it does not require availability of physical samples of thecompounds, only that their three-dimensional structure is known. It thuscan be used to design novel compounds that possess the desired abilityto inhibit telomerase.

[0067] Alternatively, this method may be used as a screening method foridentifying telomerase inhibitors from a collection of compounds thatare available, provided that the structure of these compounds is known.If only the two-dimensional structure is known, the correspondingthree-dimensional structure can be obtained using existing computerprograms. Such computer programs include but are not limited to CONCORD,CHEM3D, and MM2.

[0068] Another method for identifying G-quadruplex interactive compoundsthat may inhibit telomerase involves use of techniques such as UV/VISspectroscopy, polarimetry, CD or ORD spectroscopy, IR or Ramanspectroscopy, NMR spectroscopy, fluorescence spectroscopy, HPLC, gelelectrophoresis, capillary gel electrophoresis, dialysis, refractometry,conductometry, atomic force microscopy, polarography, dielectometry,calorimetry, solubility, EPR or mass spectroscopy. The application ofthese methods can be direct, in which the G-quadruplex interactivecompound's interaction with the G-quadruplex is measured directly, or itcan be indirect, in which a particular G-quadruplex interactive agenthaving a useful spectroscopic property is used as a probe for theability of other compounds to bind to the G-quadruplex; for example, bydisplacement or by fluorescence quenching.

[0069] III. Telomerase Inhibitors

[0070] The identification of compounds that inhibit telomerase activityprovides important benefits to efforts at treating human disease.Compounds that inhibit telomerase activity can be used to treat cancer,as cancer cells express telomerase activity and normal human somaticcells do not express telomerase activity at biologically relevant levels(i.e. at levels sufficient to maintain telomere length over many celldivisions). Unfortunately, few such compounds have been identified andcharacterized. Hence, there remains a need for compounds that act astelomerase inhibitors and for compositions and methods for treatingcancer and other diseases in telomerase activity is present abnormally.The present invention meets these and other needs.

[0071] Once a compound has been identified as being a G-quadruplexinteractive agent, confirmatory evidence for the ability of saidcompound to inhibit telomerase may be obtained using a standard primerextension assay that does not use a PCR™-based amplification of thetelomerase primer extension products such as described in Sun et al.,1997. The identified inhibitors may be used therapeutically to interferewith the function of telomerase and thus to treat cancers.

[0072] Using the screening methods described above, compounds have beenidentified that bind to human G-quadruplex structures and have beenshown to inhibit human telomerase. One group of compounds is representedby general structure I:

[0073] in which R¹ and R⁴ are represented by L-A where L is a linkinggroup which may be any of a group of substituted (X)methylene,(X)dimethylene, (X)trimethylene, (X)dimethyleneamine, (X)dimethyleneoxy,(X)dimethyleneaminodimethylene, (X)-dimethyleneoxydimethylene,(X)-p-phenylene, (X)-m-phenylene, (X)-o-phenylene, or an unsubstitutedcovalent bond;

[0074] A is a group that interacts with the grooves of the G-quadruplexstructure, examples being a substituted carbocyclic ring, a(substituted) heterocyclic ring, an hydroxyl, a carboxylic acid, acarboxylic acid ester, a carboxamide, a sulfonamide, a sulfonic acid, anitrile, a malonate diester, a malonate diamide, a disubstituted amine,a quartemized nitrogen-containing heterocyclce, or a quaternary amine.

[0075] R2, R2′, R2″, R2′″, R3, R3′, R″, R′″ are independently hydrogen,alkyl, halo, amino, nitro, hydroxy, alkoxy, alkylamino, dialkylamino,aryl, or cyano.

[0076] Another group of compounds suitable as telomerase inhibitors isshown by the general structure II in which B is O, S, or NR; C is anunsaturated linking group, 1 to 3 (substituted) ethylene groups, asubstituted or unsubstituted carbocyclic group, or a heterocyclic group;and R is lower alkyl.

[0077] These compounds may interact specifically with G-quadruplexstructures as compared to other nucleic acid structures such asdouble-stranded DNA, single-stranded DNA, and RNA structures. The degreeof selectivity in the interaction of these compounds with G-quadruplexstructures versus other nucleic acid structures is given by the ratio ofthe affinity of these compounds for G-qudruplex structures to theaffinity for the other nucleic acid structures. In particular, theability of these compounds to distinguish between G-quadruplexstructures and double-stranded DNA may be an important criterion.Compounds with general ability to bind double-stranded DNA are known toinhibit or alter a variety of DNA-associated enzymes or proteins,including but not limited to: histone binding, topoisomerase I,topoisomerase II, DNA-polymerases, RNA-polymerases, DNA repair enzymes,cytosine methyltransferase, and transcription factor binding. In orderto discover compounds that are able to selectively inhibit telomeraseand other G-qudruplex-associated enzymes and proteins, one would want toselect compounds that have a high ratio of affinities for G-qudruplexstructures versus double-stranded DNA. These selectiveG-quadruplex-binding compounds can be identified by selecting thoseG-quadruplex binding compounds that display weak or no ability forbinding to double-stranded DNA, as determined by UV/VIS spectroscopy,polarimetry, CD or ORD spectroscopy, IR or Raman spectroscopy, NMRspectroscopy, fluorescence spectroscopy, HPLC, gel electrophoresis,capillary gel electrophoresis, dialysis, refractometry, conductometry,atomic force microscopy, polarography, dielectometry, calorimetry,solubility, EPR and mass spectroscopy.

[0078] In addition to the thermodynamic considerations of G-quadruplexbinding by these compounds, the kinetics of the interaction betweenthese compounds and G-quadruplexes is also considered to be important.The relative rates of the association and dissociation of thesecompounds with G-quadruplex structures can affect their biologicalproperties. In particular, those compounds with slow dissociation ratesmay be more effective in inhibiting telomerase and otherG-quadruplex-associated enzymes and proteins than hose with identicalG-quadruplex binding ability, but whose dissociation rates are faster.For a given compound, the overall equilibrium binding affinity (bindingconstant) to G-quadruplex structures is a ratio of the association rateand the dissociation rate. The dissociation rate of a complex consistingof a G-quadruplex structure and a G-quadruplex interactive compound canbe determined by a variety of methods. In one example, the dissociationof the complex can be determined spectrophotometrically upon theaddition of a detergent, such as SDS. Alternatively, the dissociationrate can be determined in a T-jump study, in which the temperature ofthe complex is quickly raised to a point at which the complexdissociates and this process is monitored spectrophotometrically. Inanother example the dissociation rate for a G-quadruplex-compoundcomplex can be determined by monitoring by a variety of techniques thedissociation of a complex in which one partner, either the G-quadruplexor the compound, is tethered, either covalently or non-covalently, to animmobile phase, and a solution is passed over this immobilized complex.The dissociation rate for a G-quadruplex-compound complex also can bedetermined indirectly, by measuring both the equilibrium bindingconstant and the association rate. For example, if two compounds havesimilar equilibrium G-quadruplex binding constants, but one has a slowerassociation rate, then that same compound must also have aproportionately slow dissociation rate.

[0079] Using the above techniques, one can select from thoseG-quadruplex interactive agents identified, those that have additionaldesired properties of selective G-quadruplex interaction when comparedto other nucleic acids structures, such as double-stranded DNA, and/orslow kinetics of association with G-quadruplex structures.

[0080] Agents capable of inhibiting telomerase activity in tumor cellsoffer therapeutic benefits with respect to a wide variety of cancers andother conditions (for example, fungal infections) in which immortalizedcells telomerase activity are a factor in disease progression or inwhich inhibition of telomerase activity is desired for treatmentpurposes. The telomerase inhibitors of the invention can also be used toinhibit telomerase activity in germ line cells, which may be useful forcontraceptive purposes.

[0081] IV. Pharmaceutical Formulations and Administration

[0082] The invention further comprises the therapeutic treatment ofcancer by the administration of an effective dose of one or moreinhibitors of telomerase. Where clinical applications are contemplated,it will be necessary to prepare pharmaceutical compositions of drugs ina form appropriate for the intended application. Generally, this willentail preparing compositions that are essentially free of pyrogens, aswell as other impurities that could be harmful to humans or animals.

[0083] The phrase “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well know in the art. Supplementary active ingredientsalso can be incorporated into the compositions.

[0084] The active compositions of the present invention may includeclassic pharmaceutical preparations. Administration of thesecompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra. A preferred route is directintra-tumoral injection, injection into the tumor vasculature or localor regional administration relative to the tumor site.

[0085] The active compounds may also be administered parenterally orintraperitoneally.

[0086] Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0087] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifingal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

[0088] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0089] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0090] For oral administration the compounds developed in the presentinvention may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

[0091] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0092] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage could bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

[0093] Because telomerase is active only in tumor, germline, and certainstem cells of the hematopoietic system, other normal cells are notaffected by telomerase inhibition therapy Steps also can be taken toavoid contact of telomerase inhibitor with germline or stem cells,although this may not be essential. For instance, because germline cellsexpress telomerase activity, inhibition telomerase may negatively impactspermatogenesis and sperm viability, suggesting that telomeraseinhibitors may be effective contraceptives or sterilization agents. Thiscontraceptive effect may not be desired, however, by a patient receivinga telomerase inhibitor of the invention for treatment of cancer. In suchcases, one can deliver a telomerase inhibitor of the invention in amanner that ensures the inhibitor will only be produced during theperiod of therapy, such that the negative impact on germline cells isonly transient.

[0094] V. Therapies

[0095] One of the major challenges in oncology today is the effectivetreatment of a given tumor. Tumors are often resistant to traditionaltherapies. Thus, a great deal of effort is being directed at findingefficous treatment of cancer. One way of achieving this is by combiningnew drugs with the traditional therapies and is discussed below. In thecontext of the present invention, it is contemplated that therapiesdirected against telomerase could be used in conjunction with surgery,chemotherapy, radiothearpy and indeed gene therapeutic intervention. Italso may prove effective to combine telomerase targeted chemotherapywith antisense or immunotherapies directed toward tumor markers or otheroncogenes or oncoproteins.

[0096] “Effective amounts” are those amounts of a candidate substanceeffective to reproducibly decrease expression of telomerase in an assayin comparison to levels in untreated cells. An “effective amount” alsois defined as an amount that will decrease, reduce, inhibit or otherwiseabrogate the growth of a cancer cell.

[0097] It is envisioned that the telomerase inhibitors will providetherapy for a wide variety of tumors and cancers including skin cancers,connective tissue cancers, adipose cancers, breast cancers, lungcancers, stomach cancers, pancreatic cancers, ovarian cancers, cervicalcancers, uterine cancers, anogenital cancers, kidney cancers, bladdercancers, colon cancers, prostate cancers, central nervous system (CNS)cancers, retinal cancer, blood and lymphoid cancers.

[0098] A. Combination Therapies

[0099] To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, the methods of standard therapy discussed above aregenerally insufficient as tumors are often resistant to several of theseagents. Often combining a host of different treatment methods prove mosteffective in cancer therapy. Further, several AIDS afflicted patientshave a higher risk of developing cancers. Combination therapy in thesecases is required to treat AIDS as well as the cancer. Using the methodsand compounds developed in the present invention, one would generallycontact a “target” cell with a telomerase inhibitor and at least oneother agent. These compositions would be provided in a combined amounteffective to kill or inhibit proliferation of the cell. This process mayinvolve contacting the cells with the telomerase based therapy and theother agent(s) or factor(s) at the same time. This may also be achievedby contacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the telomerase based therapy and the other includesthe agent.

[0100] Alternatively, the telomerase inhibitor-based treatment mayprecede or follow the other agent treatment by intervals ranging frommin to wk. In embodiments where the other agent and telomerase-basedtherapy are applied separately to the cell, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the agent and telomerase-based treatment wouldstill be able to exert an advantageously combined effect on the cell. Insuch instances, it is contemplated that one would contact the cell withboth modalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other, with a delay time of only about 12 hbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

[0101] It also is conceivable that more than one administration ofeither telomerase-based treatment or the other agent will be desired.Various combinations may be employed, where telomerase-basedtreatment is“A” and the other agent is “B”, as exemplified below: A/B/A B/A/B B/B/AA/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/AB/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/BB/B/A/B

[0102] Other combinations are also contemplated. Again, to achieve cellkilling, both agents are delivered to a cell in a combined amounteffective to kill the cell.

[0103] The invention also encompasses the use of a combination of one ormore DNA damaging agents, whether chemotherapeutic compounds orradiotherapeutics as described in the section below, together with thetelomerase inhibitor. The invention also contemplates the use of thetelomerase inhibitors in combination with surgical removal of tumors totreat any remaining neoplastic or metastasized cells. Further,immunotherapy may be directed at tumor antigen markers that are found onthe surface of tumor cells. The invention also contemplates the use oftelomerase inhibitors in combination with gene therapy, directed towarda variety of oncogenes, such as, tumor markers, cell cycle controllinggenes, described below.

[0104] The other agent may be prepared and used as a combinedtherapeutic composition, or kit, by combining it with thetelomerase-inhibitor based treatment, as described above. The skilledartisan is directed to “Remington's Pharmaceutical Sciences” 15thEdition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

[0105] It is proposed that the regional delivery of non-nucleosideinhibitors of telomerase to patients with tumors will be a veryefficient method for delivering a therapeutically effective chemical tocounteract the clinical disease. Similarly, other chemotherapeutics,radiotherapeutics, gene therapeutic agents may be directed to aparticular, affected region of the subjects body. Alternatively,systemic delivery of telomerase based treatment and/or the agent may beappropriate in certain circumstances, for example, where extensivemetastasis has occurred.

[0106] It also should be pointed out that any of the standard or othertherapies may prove useful by themselves in treating a cancer. In thisregard, reference to chemotherapeutics and non-telomeraseinhibitor-based treatment in combination should also be read as acontemplation that these approaches may be employed separately.

[0107] When such combination therapy is employed for the treatment of atumor, the cytotoxic agent may be administered at a dosage known in theart to be effective for treating the tumor. However, the G-quadruplexinteraction compounds may produce an additive or synergistic effect witha cytotoxic agent against a particular tumor. Thus, when suchcombination antitumor therapy is used, the dosage of G-quadruplexinteraction compounds administered may be less than that administeredwhen the cytotoxic agent is used alone. Similarly, for patientsafflicted by AIDS, AZT/protease inhibitors will be used withG-quadruplex interaction compounds, or other herein mentionedtherapeutic agent(s). Again the dosage of G-quadruplex interactioncompounds or other conjunctively utilized agent, may be altered to suitthe AIDS treatment.

[0108] Preferably, the patient is treated with G-quadruplex interactioncompounds for about 1 to 14 days, preferably 4 to 14 days, prior to thebeginning of therapy with a cytotoxic agent, and thereafter, on a dailybasis during the course of such therapy. Daily treatment with thetelomerase inhibitor can be continued for a period of, for example, 1 to365 days after the last dose of the cytotoxic agent is administered.This invention encompasses the use of telomerase inhibitors-based cancertherapy for a wide variety of tumors and cancers affecting skin,connective tissues, adipose, breast, lung, stomach, pancreas, ovary,cervix, uterus, kidney, bladder, colon, prostate, anogenital, centralnervous system (CNS), retina and blood and lymph.

[0109] B. Standard Therapies

[0110] Described herein are the therapies used as standard ortraditional methods for treatment of cancers. The section onchemotherapy describes the use of non-nucleoside telomerase inhibitorsas chemotherapeutic agents in addition to several other well knownchemotherapeuticagents. As detailed in the section above, all themethods described below can be used in combination with the telomeraseinhibitors developed in the present invention.

[0111] a. Surgery: Surgical treatment for removal of the cancerousgrowth is generally a standard procedure for the treatment of tumors andcancers. This attempts to remove the entire cancerous growth. However,surgery is generally combined with chemotherapy and/or radiotherapy toensure the destruction of any remaining neoplastic or malignant cells.

[0112] b. Chemotherapy: A variety of chemical compounds, also describedas “chemotherapeutic agents”, function to induce DNA damage, are used totreat tumors. Chemotherapeutic agents contemplated to be of use,include, adriamycin, 5-fluorouracil (5FU), etoposide (VP-16),camptothecin, actinomycin-D, mitomycin, cisplatin (CDDP), hydrogenperoxide, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, tamoxifen, taxol,transplatinum, vincristin, vinblastin and methotrexate to mention a few.

[0113] Agents that damage DNA include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

[0114] Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. A number of such agentshave been developed, particularly useful are agents that have undergoneextensive testing and are readily available. 5-fluorouracil (5-FU), isone such agent that is preferentially used by neoplastic tissue, makingit particularly useful for targeting neoplastic cells. Thus, althoughquite toxic, 5-FU, is applicable with a wide range of carriers,including topical and even intravenous administrations with dosesranging from 3 to 15 mg/kg/day.

[0115] Agents that directly cross-link nucleic acids, specifically DNA,are envisaged to facilitate DNA damage leading to a usefulantineoplastic treatment. For example, cisplatin, and other DNAalkylating agents may be used. Cisplatin has been widely used to treatcancer, with efficacious doses used in clinical applications of 20 mg/m²for 5 days every three wk for a total of three courses. Cisplatin is notabsorbed orally and must therefore be delivered via injectionintravenously, subcutaneously, intratumorally or intraperitoneally.

[0116] The non-nucleoside G-quadruplex inhibitor compounds developed inthis invention are chemotherapeutic agents that are cytotoxic andinihibit telomerase function which is critical to cell replication andmaintenance of tumor cell immortality. These compounds also indirectlyinhibit DNA polymerases by their strong interactions with theG-quadruplex structures (FIG. 7).

[0117] c. Radiotherapy: Radiotherapeutic agents and factors includeradiation and waves that induce DNA damage for example, y-irradiation,X-rays, UV-irradiation, microwaves, electronic emissions, radioisotopes,and the like. Therapy may be achieved by irradiating the localized tumorsite with the above described forms of radiation's. It is most likelythat all of these factors effect a broad range of damage DNA, on theprecursors of DNA, the replication and repair of DNA, and the assemblyand maintenance of chromosomes.

[0118] Dosage ranges for X-rays range from daily doses of 50 to 200roentgens for prolonged periods of time (3 to 4 wk), to single doses of2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, anddepend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

[0119] d. Gene Therapy: Gene therapy based treatments targeted towardsoncogenes such as p53, p16, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA 1, VHL, FCC, MCC, ras, myc, neu, raferb, src, fins, jun, trk; ret, gsp, hst, bcl and abl, which are oftenmutated versions of their normal cellular counterparts in canceroustissues.

[0120] VI. Screening for Anti-Telomere and Anti-Cancer Activity

[0121] In particular embodiments, one may test the inhibitors bymeasuring their ability to inhibit growth of cancer cells, to inducecytotoxic events in cancer cells, to induce apoptosis of the cancercells, to reduce tumor burden and to inhibit metastases. For example,one can measure cell growth according to the MTT assay. A significantinhibition in growth is represented by decreases of at least about30%40% as compared to uninhibited, and most preferably, of at leastabout 50%, with more significant decreases also being possible. Growthassays as measured by the MTT assay are well known in the art. Otherassays to measure cell death, apoptosis are well known in the art, forexample, Mosmann et al., 1983; Rubinstein et al., 1990.

[0122] Quantitative in vitro testing of the anti-tumor agents identifiedherein is not a requirement of the invention as it is generallyenvisioned that the agents will often be selected on the basis of theirknown properties or by structural and/or functional comparison to thoseagents already demonstrated to be effective. Therefore, the effectiveamounts will often be those amounts proposed to be safe foradministration to animals in another context.

VII. EXAMPLES

[0123] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Telomerase Inhibition byN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide

[0124] Selection ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide as a potential telomerase inhibitor was determined by Method (B)as described in Example 2.

[0125] The relative inhibition of telomerase byN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide was determined in a standard primer extension assay that doesnot use a PCR™-based amplification of the telomerase primer extensionproducts. Briefly, the 18-mer telomeric primer d[TTAGGG]₃ (1 μM) withoutor with N,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylicacid diimide was elongated with telomerase in the presence of 1.5 μM of[α-³²P]-dGTP (800 Ci mmol⁻¹, 10 mCi ml⁻¹) with 1 mM dATP and 1 mM dTTP.The extension products were isolated and visualized by autoradiographyafter denaturing gel electrophoresis.

[0126] The IC₅₀ was determined to be 50 μM, and at 100 μM ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide there is an almost complete inhibition of telomerase activity.N,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimideand 3 showed similar behavior.

Example 2 Screening Assays for Telomerase Inhibitors

[0127] Compounds that inhibit telomerase are potential drugs for thetreatment of cancer. The method selects compounds based upon ability tointeract with the human DNA-G-quadruplex. Several procedures fordetecting this interaction include:

[0128] (A) A three dimensional structure of a candidate compound will beanalyzed to determine their degree of complementarity to thethree-dimensional structure of human telomeric DNA G-quadruplex. The NMRsolution structure of d(AGGGTTAGGGTTAGGGTTAGGG) [pdb entry 143d] and itscorresponding molecular surface, generated with the ms program, wereused as inputs to the SPHGEN program. The resulting sphere cluster wasused as input to DOCKv2.0 and a subset of the Cambridge crystallographicdatabase was search using the contact scoring algorithm.N,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide was found to have one of the highest contact scores in the ˜2000compounds examined.

[0129] (B) Compounds may be selected for their ability to interact withhuman DNA G-quadruplex as indicated by UV/VIS spectroscopy. To a 10 μMsolution ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide in 20 mM phosphate buffer containing 100 mM KCl, pH 7.0 in aquartz cuvette was added 10 μL aliquots of a 3 mM solution ofd(TTAGGGT)₄. After each addition the UV/VIS spectrum was recorded.Pronounced changes in the UV/VIS spectrum of the compound were noted atwavelengths 488 nm (˜40% hypochromicity), 510 nm ˜50% hyperchromicity),and 548 nm (˜200% hyperchromicity).

[0130] (C) Compounds may be selected for their ability to interact withhuman DNA G-quadruplex as indicated by NMR spectroscopy. The iminoproton spectrum (9-12 ppm) of a solution of d(TTAGGG)4 in D2O/H2O(10:90) was determined at 500 MHz. Aliquot ofN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimidewere added and the imino proton spectrum recorded. At an overallstoichiometry of 1:1 the G6 imino resonance becomes significantlybroader and shifts >0.2 ppm upfield.

[0131] (D) Compounds may be selected for their ability to interact withhuman DNA g-quadruplex as indicated by an increase in the meltingtemperature of the G-quadruplex structure. Thermal denaturation of theparallel four-stranded G-quadruplex structure formed by the d[T₂AG₃T](7-mer) (125 mM KCl, 25 mM KH₂PO₄, 1 mM EDTA, pH 6.9) monitored by NMR.The spectrum for DNA alone and in the presence ofN,N′-bis(2-dimethylaminooethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide. The molar ratio ofN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide to quadruplex was 4:1. The imino proton signals have beenassigned previously (Laughlan et al., 1994) as G6, G5, and G4 from highto low field. The presence of drug leads to line broadening and anupfield shift of the imino proton signals indicative of intercalation.Furthermore, the melting temperature of the DNA G-quadruplex isincreased significantly in the presence of the compound. Spectra wereacquired in 90% H₂O/10% D₂O on a Bruker AMX 500 MHz spectrometer atvarious 15-85° C. using a 1-1 echo pulse sequence with a maximumexcitation centered at 12.0 ppm. A total of 128 scans was obtained foreach spectrum with a relaxation delay of 2 s. Before acquiring thespectrum at each temperature, the sample was allowed to equilibrate atthe new temperature for at least 10 min. The data were processed with anexponential window function using 2 Hz of line broadening. The dataindicate thatN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide increases the melting temperature of the G-quadruplex by atleast 20° C.

Example 3 Synthesis ofN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic acid diimide

[0132] N,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide was prepared by mixing three g of3,4,9,10-perylenetetracarboxylic acid dianhydride with 2.5 mL of1-(2-aminoethyl)piperidine in 10 mL of DMA and 10 mL of 1,4-dioxane. Themixture was heated under reflux for 6 hours, and the solvents removedunder reduced pressure. The residue was dissolved in −100 mL ofdistilled water, and insoluble components were removed by filtration.The pH of the resulting solution was adjusted to −3 with the addition ofHCl, and the solution was allowed to stand overnight. Precipitatedimpurities was removed by filtration, and the resulting solution wasadjusted to pH 1-12 with the addition of NaOH. The precipitated productwas isolated by filtration, washed with water and dried under vacuum.

Example 4 DNA Synthesis Arrest Assay

[0133] It has been shown that DNA sequences with quadruplex-formingpotential present obstacles to DNA synthesis by DNA polymerases in a K⁺dependent manner. This K⁺ dependent block to DNA polymerase is aselective and sensitive indicator of the formation of intramolecularquadruplexes (Weitzmann, et al., 1996). This assay has been adapted todemonstrate the stabilization of quadruplex by small molecules and usedto screen potential G-quadruplex-interactive compounds.

[0134] The assay is a modification of that described by Weitzmann, etal. Briefly, primers (24 nM, sequence: 5′-TAATACGACTCACTATAG-3′) labeledwith [y-³²P]ATP were mixed with template DNA PQ74(12 nM,

[0135] sequence:TCCAACTATGTATACTTGGGGTTGGGGTTGGGG

[0136] TTGGGGTTGGGGTTAGCGGCACGCAATTGCTATAGTGAGTCGTATTA-3′) in a Tris-HClbuffer (10 mM Tris, pH8.0) containing 5 mM K⁺ and heated at 90° C. for 4min. After cooling at room temperature for 15 min. potentialG-quadruplex-interactive compounds were then added to variousconcentrations. The primer extension reactions were initiated by addingdNTP (final concentration 100 μM), MgCl₂ (final concentration 3 mM) andTaq polymerase (2.5 U/reaction, Boehringer Mannheim). The reactions wereincubated at 55° C. for 15 min. then stopped by adding an equal volumeof stop buffer (95% formamide, 1 mM EDTA, 10 mM NaOH, 0.1% xylenecyanol. 0.1% bromophenol blue). The products were separated on a 12%polyacrylamide sequencing gel. The gels were then dried and visualizedon a phosphorimager (Molecular Dynamics model 445 S1).

[0137] To validate the assay, G-quadruplex interactive compounds such asporphyrins and perylenes were tested. The results were consistent withNMR and telomerase inhibition data. FIG. 8 shows the DNA synthesisarrest induced by Quinobenzoxazine analogs (QQ23, 1130, QQ31) and aperylene compound (APPER).

Example 5 Photocleavage Assay to Detect Quadruplex DNA Interactions

[0138] (i) Design and Synthesis of an Intramolecular Quadruplex DNA

[0139] The oligonucleotide G4A employed was synthesized on a PerseptiveDNA synthesizer and deprotected following the routine phosporamiditeprocedures the DNA was purified by polyacrylamide gel electrophoresis(PAGE). The sequence for this 39 oligomer single strand DNA is:5′ CATGGTGGTTTGGGTTAGGGTTAGGGTTAGGGTTACCAC 3′.

[0140] This human telomere repeat-containing DNA was designed to form anintramolecular quadruplex which can be stabilized by the stem region in(FIG. 5). A sticky end was added so that unusual secondary structurescould be detected by ligation assay once they are formed.

[0141] (ii) Photocleavage Assay

[0142] The G4A DNA was labeled with ³²P at the 5′ end and stored in 1×TEbuffer at 3000 cpm/μl. For each photocleavage reaction, 10μ of DNA (˜5ng) was mixed with 10 μl of 200 mM KCl or 200 mM NaCl and boiled for 10min before cooled down to room temperature. For the no porphyrin controlsamples, 10 μl of distilled water was added instead. The mixtures weretransferred to a 96 well plate and added with 2 μl of 1 μM TMPyP⁴aqueous solution. The samples were then exposed to 24 watts fluorescentdaylight under a glass filter for different periods of time. Then thereactions were stopped with 100 μl of calf thymus DNA (0.1 μg/μl). Afterphenol-chloroform extraction, the samples were subjected to strandbreakage treatment and ethanol precipitation. The DNA samples wereloaded onto a 16% polyacrylamide gel for electrophoresis and visualizedwith PhosphorImager (from Molecular Dynamics, Inc.). A typical resultfor the photocleavage assay is shown in FIG. 6.

Example 6 Selection of G-Quadruplex Selective Ligand

[0143] The N,N′-bis(3-morpholinopropyl)3,4,9,10-perylenetetracarboxilicacid diimide (KeTEL01) was synthesized from3,4,9,10-perylenetetracarboxylic acid dianhydride and3-morpholinopropylamine using a procedure analogous to that describedabove in example 5.3 for the synthesis ofN,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide. A solution of KeTEL01 was prepared by dissolving 1 mg ofKeTEL01 in 300 μL of 1N HCl. To this solution was added 11 mL of a pH7.0 buffer containing 20 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, and0.02% hydroxypropyl-β-cyclodextrin. Aliquots of this stock solution ofKeTEL01 were transferred to 8 different quartz cuvettes and diluted intopH 7.0 mM sodium phosphate, 100 mM KCl, 1 mM EDTA buffer to affordsolutions in which the concentration of KeTEL01 was 20 μM. To each ofthe cuvettes was added a solution of [d(TTAGGGT)]4 so that the finalconcentration of [d(TTAGGGT)]4 in each of the cuvettes was 0, 4, 8, 12,16, 20, 50, and 80 μM. These solutions were allowed to stand overnightin the dark, and the UV/VIS spectrum of each was determined. Pronounced,G-quadruplex concentration-dependent changes in the UV/VIS spectrum werenoted at wavelengths 488 nm (˜40% hypochromicity), 510 nm (˜40%hyperchromicity) and 548 nm (˜100% hyperchromicity). In a parallelstudy, changes in the UV/VIS spectrum of a 20 μM solution o f KeTEL01 ina pH 7.0 20 mM phosphate buffer containing 100 mM KCl and 1 mM EDTA weredetermined upon the addition of 10 μM aliquots of a 3 mM (base pair)solution of calf thymus DNA. No changes in the UV/VIS spectrum of thissolution were noted, indicating that KeTEL01 does not interact withdouble-stranded DNA.

Example 7 Kinetics of Interaction of KeTEL01 with G-Quadruplex DNA

[0144] A solution of 5 μM KeTEL01 in 20 mM phosphate buffer, 100 mM KCl,pH 7.0 was placed in a quartz cuvette and the UV/VIS spectrumdetermined. An aliquot of a solution of [d(TTAGGGT)]4 was added to thecuvette to afford a final concentration of 50 μM. The cuvette wasquickly inverted several times and placed in the spectrophotometer. Theabsorption of the sample at 488 nm was continuously monitored for 3hours, during which time, the absorption decreased in a multiexponentialfunction. The time required for the absorption at 488 nm to reachone-half of its equilibrium value was 60 min.

Example 8 Telomerase Inhibition by UT-SK-02 (DiethylthiocarbocyanineIodide)

[0145] Using the DOCK screening methods above, the carbocyanine group ofcompounds were identified as potential G-quadruplex interactive agents.A number of these compounds were assayed using the DNA Synthesis ArrestAssay described in example 5.4. Each compound was assayed at aconcentration of 20 μM. The results of this study are summarized inTable 1 ahead: TABLE 1 Stop Stop Compound (F + P)_(rel)* (P/T)_(rel)**IBT-129A 68% 91% UT-SK-001 86% 83% UT-SK-002 88% 139%  UT-SK-003 29% 32%UT-SK-004 71% 88% UT-SK-006 33% 12%

[0146] Of the compounds tested, only one, UT-SK-002(diethylthiocarbocyanine iodide) demonstrated a specific interactionwith G-quadruplex DNA, as indicated by a relative ratio of paused tototal DNA product greater than 100% and a relative amount of DNAproducts, both paused and full-length, that is close to 100%. Inconfirmatory tests, only this compound inhibited telomerase, with aninhibition of 10-35% at a concentration of 50 μM.

Example 9 Reduced Cellular Proliferation by Selected Compounds

[0147] The ability of these compounds to inhibit the proliferativecapacity of human cancer cells was determined by a standard MTT assay.Briefly, cells were incubated for 72 hours in the presence of variousconcentrations of compound, and the cell viability was determined bymonitoring the formation of a colored formazan salt of the tetrazoliumsalt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)by viable cells. KeTEL01 showed no cytotoxic effect up to the highestconcentrations tested (100 μM), whereas KeTEL03(N,N′-bis(2-dimethylaminoethyl)-3,4,90,10-perylenetetracarboxylica aciddiimide, see Example 1) showed good cytotoxicity in a variety of humancancer cell lines. Thus, KeTEL01, which is a selective G-quadruplexinteractive agent, has no acute (72 hr) cytotoxic effect, but thestructurally analogous KeTEL03, which does interact with double-strandedDNA as well as G-quadruplex, is cytotoxic under these assay conditions.Cytotoxicity - IC₅₀ MTT Cell Lines KeTEL01 KeTEL03 MCF-7 >100 μM 18.4μM  BT-20 >100 μM 3.8 μM PC-3 >100 μM 3.8 μM Raji >100 μM 0.4 μM

Example 10 DNA Oligonucleotides

[0148] The DNA primer extension sequence P18 (5′-TAATACGACTCACTATAG-3′)and the template sequences shown in Table 1 were synthesized using aPerSeptive Biosystems Expedite 8909 synthesizer and purified withdenaturing polyacrylamide gels. The template DNA was diluted to 5 ng/μLand dispensed into small aliquots.

Example 11 DMS Methylation Protection Assay

[0149] The ³²P-labeled PQ74 and HT4 templates were denatured by heatingat 90° C. for 5 min and then cooled down to room temperature in 50 mMTris-HCl buffer with or without 100 mM of K⁺. One microliter of 1:4ethanol-diluted DMS was added to 1 μg (300 μL) of annealed DNA. Aliquotswere taken at time points as indicated in the figures, and modificationreactions were stopped by adding ¼ volume of stop buffer containing 1Mof β-mercaptoethanol and 1.5 M of sodium acetate. The modificationproducts were ethanol precipitated twice and treated with piperidine.After ethanol precipitation, the cleaved products were resolved on a 16%polyacrylamide gel.

Example 12 DNA Synthesis Arrest Assay

[0150] This assay is a modification of that described by Weitzmann andco-workers (Weitzmann et al., 1996). Briefly, primers (P18, 24 nM)labeled with [γ-³²P] were mixed with template DNA (12 nM)) in a Tris-HClbuffer (10 mM Tris, pH 8.0) containing K⁺ (5 mM for the PQ74 templateand 50 mM for the HT4 template) and denatured by heating at 90° C. for 5min. After cooling down to room temperature, BSU-1051 was added atvarious concentrations and incubated at room temperature for 15 min. Theprimer extension reactions were initiated by adding dNTP (finalconcentration 100 μM), MgCl₂ (final concentration 3 μM), and Taq DNApolymerase (2.5 U/reaction, Boehringer Mannheim). For sequencingreactions, the TaqTrack Sequencing System (Promega, Wis., Madison) wasused. The sequencing reaction buffer was changed to 50 mM Tris-HCl, pH9.0, 10 mM MgCl, and 50 mM K⁺. The reactions were stopped by adding anequal volume of stop buffer (95% formamide, 10 mM EDTA, 10 mM NaOH, 0.1%xylene cyanol, 0.1% bromphenol blue). For the temperature-dependentstudies, the ligand concentration was fixed and the primer extensionreactions were carried out at the temperatures indicated in methods. Theproducts were separated on a 12% polyacrylamide sequencing gel. The gelswere then dried and visualized on a PhosphorImager (Molecular Dynamicsmodel 445 S1).

Example 13 Results

[0151] (i) The G-rich Regions of the PQ74 and HT-4 Templates FormIntermolecular G-quadruplex Structures In K⁺ Buffer

[0152] To determine the nature of the G-quadruplex structures formed bythe template sequences used in this study (see Table 1), dimethylsulfate(DMS) was used to probe the accessibility of N7 of guanine in the DNAtemplates (Maxam and Gilbert, 1980). When the PQ74 template wasmethylated in 1×TE buffer, there was no apparent protection of anyguanine N7. However, with the exception of the first guanine in each ofthe four TTGGGG repeats, all the guanines in the G-rich region of thePQ74 template are protected from reacting with DMS in 100 mM K⁺ buffer,whereas guanines located outside the four repeats react strongly withDMS. This DMS protection pattern for the G-rich region of the PQ74template in K⁺ buffer suggests that only three guanines in each of thefour TTGGGG repeats are involved in G-tetrad formation. This DMSreaction pattern is different from that observed previously by Hendersonand co-workers (Henderson et al., 1990) with the d(TTGGGG)₄ G-quadruplexin which only the first guanine of the third repeat (corresponding to G9in the PQ74 template) is hypersensitive to DMS methylation. On the basisof the results from the inventors' study, they propose a model for theG-quadruplex structure formed by the G-rich region of the PQ74 sequenceconsisting of d(TTGGGG)₄. In this model, the first guanine of the firstrepeat is located in the 5′ overhang region and is therefore open to DMSmethylation. However, the first guanines of the second, third, andfourth repeats (G5, G9, and G13, respectively) are located in the loopregions of the G-quadruplex. Although the N7 groups of these three loopguanines are not involved in hydrogen bonds, steric inaccessibility mayprotect them from DMS methylation. The DMS footprinting pattern showsthat while they are partially protected from DMS methylation, thisprotection is less than that for the other guanines in the repeat.

[0153] The TTAGGG repeats in the G-rich region of the HT-4 template alsoshowed high DMS methylation protection in K⁺ buffer. In this particularcase, all three guanines in each repeat were almost evenly protectedfrom methylation, indicating that all of them are involved in G-tetradformation. This DMS methylation pattern is consistent with theintramolecular G-quadruplex structure proposed by Patel and co-workersfor the d[AG₃(T₂AG₃)₃] sequence based on NMR studies (Wang and Patel,1993).

[0154] (ii) BSU-1051 Binds to G-quadruplex DNA and Blocks DNA SynthesisIn a Concentration Dependent Manner

[0155] Although it has been shown that G-quadruplex structures blockprimer extension by DNA polymerase in a K⁺ dependent manner (Weitzmannet al., 1996), the inventors are unaware of any reports showing enhancedblockage by G-quadruplex—interactive agents. To determine if BSU-1051binding to G-quadruplex enhances the block to DNA synthesis, primerextension reactions were carried out in the absence and presence ofBSU-1051. Taq DNA polymerase primer extension on DNA templatescontaining four repeats of either TTGGGG (PQ74) or TTAGGG (HT4) in thepresence of different concentrations of BSU-1051 at 55° C. wereperformed. In these studies, K⁺ was added at low concentrations (5 mM ofK⁺ for the PQ74 template and 20 mM of K⁺ for the HT4 template) in orderto prevent overwhelming polymerase pausing due to formation of highlystable G-quadruplex structures. In the absence of BSU-1051, there isonly a slight pausing of the Taq DNA polymerase when it reaches the3′-end of the G-rich site on the template DNA at 55° C. However, uponincreasing the concentration of BSU-1051, enhanced pausing is observedat the same site as that seen with low K⁺ concentrations. This suggeststhat BSU-1051 enhances the polymerase pausing by stabilizing theG-quadruplex structure formed in the K⁺ buffer. At high BSU-1051concentrations, the inventors not only observed enhanced pausing at the3′-end of the G-quadruplex site but also increased premature terminationresulting from nonspecific interactions between BSU-1051 and thesingle-stranded template DNA. At a BSU-1051 concentration of 100 μM, theprimer extension is completely inhibited due presumably to nonspecificinteractions between BSU-1051 and the single- and/or double-stranded DNAor between BSU-1051 and the polymerase itself. In addition to theprimary pausing site at the beginning of the G-quadruplex site, twoother secondary pausing sites at the second and third G-rich repeats areobserved at high BSU-1051 concentrations. These pausings are probablyinduced by other structures formed by this G-rich sequence. Given thefact that secondary pausing beyond the first G-tetrad is not seen in thesequencing lanes that contain 50 mM K⁺, it is likely that thesesecondary pausings are caused by hairpin structures that are stabilizedby BSU-1051 but not K⁺. This suggests that BSU-1051 has a relativelyhigher affinity for G-quadruplex DNA over other DNA secondary structuresor single- and double-stranded DNA.

[0156] (iii) DNA Synthesis Arrest by the BSU-1051-Quadruplex ComplexDepends On the Stability of the G-quadruplex Structure

[0157] To further evaluate the ability of BSU-1051 to stabilizeG-quadruplex DNA, Taq DNA polymerase primer extension reactions werecarried out at five different temperatures in the presence and absenceof BSU-1051. In the absence of BSU-1051 polymerase pausing on the PQ74template containing four repeats of TTGGGG is almost lost at around 65°C., which is presumably the melting point of the G-quadruplex structureformed by this G-rich region in the template DNA. On the other hand, inthe presence of 20 μM BSU-1051, the G-quadruplex structure is furtherstabilized, and significant pausing is observed up to 74° C. In the HT4template containing four repeats of TTAGGG, in which the G-quadruplexstructure formed is presumably less stable, pausing fades out at 55° C.in the absence of the ligand. However, in the presence of BSU-1051,pausing is observed up to 65° C. Thus, for both DNA sequences, ΔTM uponthe addition of 20 μM BSU-1051 is about 20° C.

[0158] In order to confirm that the pausings seen result from theformation of a G-quadruplex structure on the template DNA, certainguanines in the templates were substituted with 7-deaza-dG. Since N7 ofguanine is involved in hydrogen bonding in the formation of aG-quadruplex structure, substitution of guanine with 7-deaza-dG shouldpreclude the formation of any G-quadruplex structure and allow foruninterrupted primer extension on the template by Taq DNA polymerase inthe presence of either K⁺ or BSU-1051. As shown in Table 1, two guaninesin the TTAGGG repeat region of the HT4 template and four guanines in theTTGGGG repeat region of the PQ74 template were replaced with 7-deaza-dG.This change would allow the formation of no more than two intramolecularG-tetrads and should lead to destabilization of the intramolecularG-quadruplex structure. The primer extension results with these7-deaza-dG substituted templates indicate that no significant pausingoccurs in either template in the presence of up to 20 mM of K⁺ or atBSU-1051 concentrations of up to 50 μM. This result provides strongsupport for the conclusion that BSU-1051 binds to and stabilizesintramolecular G-quadruplex DNA, leading to pronounced DNA synthesisarrest at the G-quadruplex site in the original G-rich templates.

Example 14 Discussion

[0159] G-rich sequences such as telomeric DNA and triplet DNA have beenreported to form parallel or antiparallel G-quadruplex structures in thepresence of monovalent cations such as Na⁺ and K⁺. Williamson andco-workers observed very strong intramolecular UV cross-linking for thesequence d(TTGGGG)₄ in a 50 mM K⁺ buffer (Williamson et al., 1989).Their results indicate that this sequence forms an intramolecularstructure. Using DMS methylation, the inventors conclude that fourrepeats of TTGGGG or TTAGGG within a non-G-rich sequence are capable offorming an intramolecular G-quadruplex structure in K⁺ buffer.Furthermore, the DMS methylation results indicate that of the possibletypes of G-quadruplex structures that could be formed by d(TTGGGG)₄, astructure consisting of three G-tetrads is the predominant species in100 mM of K⁺ buffer. The proposed G-quadruplex structures formed byd(TTGGGG)₄ and d(TTAGGG)₄ repeats have diagonal loops, but alternativeintramolecular G-quadruplex structures formed by foldover hairpinsconsisting of three G-tetrads are also possible (Williamson, 1994; Wangand Patel, 1995; Wang and Patel, 1994). However, the inventors could notdifferentiate between these two different types of intramolecularG-quadruplex structures by the DMS methylation pattern alone.

[0160] G-rich sequences that are capable of forming G-quadruplexes invitro can be found in telomeric sequences (Blackburn, 1991; Sundquistand Klug, 1989; Kang et al., 1992), immunoglobulin switch regions (Senand Gilbert, 1988), the insulin gene (Hommond-Kosack et al., 1993), thecontrol region of the retinoblastoma susceptibility gene (Murchie andLilley, 1992), the promoter region of c-myc gene (Simonsson et al.,1998), fragile X syndrome triplet repeats (Nadel et al., 1995; Fry andLoeb, 1994), and HIV-1 RNA (Awang and Sen, 1993). It has been suggestedby Sen and Gilbert that telomeric DNA sequences may associate toinitiate the alignment of four sister chromatids by forming parallelguanine quadruplexes (Sen and Gilbert, 1990). Furthermore, the discoveryof G-quadruplex-forming sequences in the promoter region of certaingenes suggests that G-quadruplex structures may play a role in thetranscription regulation of these genes. Another possible role ofG-quadruplex DNA is the regulation of telomere length, since a telomericoverhang that forms a G-quadruplex structure would not be a goodsubstrate for telomerase (Henderson and Blackburn, 1989; Zahler et al.,1991). The inventors recently have demonstrated that BSU-1051 inhibitsprimer extension by telomerase only when the substrate (telomeric DNA)reaches four or more repeats in length (Sun et al., 1997). In thisreport, the inventors show that BSU-1051 is able to bind to andstabilize the intramolecular G-quadruplex structure formed by fourtelomeric repeats. Thus, it is reasonable to postulate that BSU-1051inhibits telomerase by interacting with its substrate(G-quadruplex-forming telomeric repeats) rather than telomerase itself.If G-quadruplex structures play important roles in other biologicalprocesses, then G-quadruplex-interactive compounds such as thosedescribed here, which stabilize these structures, may have a variety ofbiological effects. A series of 2,6-diamidoanthraquinones, includingBSU-1051, has been reported to moderate conventional cytotoxicity in arange of tumor cells (Collier and Neidle, 1988; Agbandje et al., 1992)and to inhibit human telomerase (Perry et al., 1998). The G-quadruplexbinding property of those compounds provides a possible mechanism fortheir action, although other mechanisms involving targeting of duplexDNA are also likely.

[0161] The inventors have recently proposed a model for aperylene—quadruplex complex based on NMR evidence (Fedoroff et al.,1998). By analogy with this structure and that proposed for aTMPyP₄—G-quadruplex structure (Wheelhouse et al., 1998), it seems mostlikely that the binding site of the BSU-1051 is external to the lowerG-tetrad and within the diagonal loop (see FIG. 5).

[0162] The block of DNA synthesis by G-quadruplex structures is notpolymerase specific. Woodford and co-workers showed that the K⁺dependent DNA synthesis arrest by G-quadruplex structures is similar forvarious polymerases (Woodword et al., 1994). The inventors have foundthat the BSU-1051—induced DNA synthesis arrest pattern is virtuallyidentical when Taq DNA polymerase, E. coli DNA polymerase I (Klenowfragment), or AMV reverse transcriptase is used. Given the fact thatmany G-rich DNA sequences are capable of forming G-quadruplexes in-vitro(particularly some cancer related genes and sequences such as c-myc andtelomeres), G-quadruplexs are targets for anticancer chemotherapy. TheDNA synthesis stop assay described in this report provides a simple andrapid method for the identification of G-quadruplex—interactive agentsas lead compounds. This polymerase stop assay also allows an internalcomparison for the relative binding of potentialG-quadruplex—interactive compounds with single and double-stranded DNAtargets. This is an important comparison that may provide clues as tothe relative cytotoxicity of these compounds.

[0163] The inventors have successfully used the present assay in theidentification and characterization of other G-quadruplex—interactivecompounds that are also telomerase inhibitors (Fedoroff et al, 1998;Wheelhouse et al., 1998). This assay can be used to identify otherG-quadruplex—interactive compounds with potential clinical utility.

REFERENCES

[0164] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0165] Agbandje, Jenkins, McKerma, Reszka, Neidle,“Anthracene-9,10-diones as potential anticancer agents. Synthesis, DNAbinding, and biological studies on a series of 2,6-disubstitutedderivatives,” Med. Chem., 35:1418-1429, 1992.

[0166] Allshire, Gosden, Cross, Cranston, Rout, Sugawara, Szostak,Fantes, Hastie, “Telomeric repeat from T. thermophilia cross hybridizeswith human telomeres,” Nature, 332:656-659, 1988.

[0167] Blackburn, “Structure and function of telomeres,” Nature,350:569-573, 1991.

[0168] Blackburn, Greider, Eds., In: Telomeres,” Cold Spring HarborPress, New York, 1995.

[0169] Broccoli, Young, de Lange, “Telomerase activity in normal andmalignant hematopoietic cells,” Proc. Natl. Acad. Sci. U.S.A,92:9082-9086, 1994.

[0170] Chen, Kuntz, Shafer, “Spectroscopic recognition of guaninedimeric hairpin quadruplexes by a carbocyanine dye,” Proc. Natl. Acad.Sci. U.S.A., 93:2635-2639, 1996.

[0171] Collier and Neidle, “Synthesis, molecular modeling, DNA binding,and antitumor properties of some substituted amidoanthraquinones,” Med.Chem., 31:847-857, 1988.

[0172] Collins and Greider, “Tetrapymena telomerase catalyzesnucleolytic cleavage and nonprocessive elongation,” Genes Dev.,7:1364-1376, 1993.

[0173] Counter, Avilion, LeFeuvre, Stewart, Greider, Harley, Bacchetti,“Telomere shortening associated with chromosome instability is arrestedin immortal cells which express telomerase activity,” EMBO J.,11:1921-1929, 1992.

[0174] Feng, Funk, Wang, Weinrich, Avilion, Chin, Adams, Chang, Allsopp,Yu, Le, West, Harley, Andrews, Greider, Villeponteau, “The RNA componentof human telomerase,” Science, 269:1236-1241, 1995.

[0175] Fox, Polucci, Jenkins, Neidle, “A molecular anchor forstabilizing triple-helical DNA,” Proc. Nail. Acad. Sci. USA.,92:7887-7891, 1995.

[0176] Haq, Ladbury, Chowdry, Jenkins, “Molecular anchoring of duplexand triplex DNA by disubstituted anthracene-9/10-diones: calorimetric,UV melting, and competition dialysis studies,” J. Am. Chem. Soc.,118:10693-10701, 1996.

[0177] Harley, Futcher, Greider, “Telomeres shorten during aging ofhuman fibroblasts,” Nature, 345:458460, 1990.

[0178] Harley, Kim, Prowse, Weinrich, Hirsch, West, Bacchetti, Hirte,Counter, Greider, Wright, Shay, “Telomerase, Cell Immortality, andCancer,” Cold Spring Harbor Syrup. Quant. Biol., 59:307-315, 1994.

[0179] Hiyama, Hiyama, Ishioka, Yamakido, Inai, Gazdar, Piatyszek, Shay,“Telomerase activity in small-cell and non-small-cell lung cancers,”Natl. Cancer Inst., 87:895-902, 1995a.

[0180] Hiyama, Hiyama, Yokoyama, Matsuura, Piatyszek, Shay, “Correlatingtelomerase activity levels with human neuroblastoma outcomes,” NatureMedicine, 1:249-255, 1995a.

[0181] Kang, Zhang, Ratlift, Moyzis, Rich, “Crystal structure offour-stranded Oxytricha telomeric DNA,” Nature, 356:126-131, 1992.

[0182] Kim, Piatyszek, Prowse, Harley, West, Ho, Coviello, Wright,Weinrich, Shay, “Specific association of human telomerase activity withimmortal cells and cancer,” Science, 266:2011-2015, 1994.

[0183] Laughlan, Murchie, Norman, Moore, Moody, Lilley, Luisi, “Thehigh-resolution crystal structure of a parallel-stranded guaninetetraplex,” Science, 265:520-524, 1994.

[0184] Norton, Piatyszek, Woodring, Shay, Corey, “Inhibition of humantelomerase activity by peptide nucleic adds,” Nature Biotechnology,14:615-619, 1996.

[0185] Parkinson, “Do telomerase antagonists represent a novelanti-cancer strategy?” Brit. J Cancer, 73:1-4, 1996.

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[0188] Tanious, Jenkins, Neidle, Wilson, “Substituent position dictatesthe intercalative DNA-binding mode for anthracene-9,10-dione antitumordrugs,” Biochemistry, 31:11632-11640, 1992.

[0189] Wang and Patel, “Guanine residues in d(T₂AG₃) and d(T₂G₄) formparallel-stranded potassium cation stabilized G-quadruplexes with antiglycosialic torsion angles in solution, Biochemistry, 31:8112-8119,1992.

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1 12 1 6 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 1 ttaggg 6 2 22 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 2 agggttaggg ttagggttag gg 22 3 7DNA Artificial Sequence Description of Artificial Sequence SyntheticPrimer 3 taagggt 7 4 8 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 4 ttagggtt 8 5 7 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 5 aatgggt 7 6 7 DNAArtificial Sequence Description of Artificial Sequence Synthetic Primer6 ttagggt 7 7 6 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 7 ttgggg 6 8 22 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 8 agggttagggttagggttag gg 22 9 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 9 taatacgact cactatag 18 10 80 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 10tccaactatg tatacttggg gttggggttg gggttggggt tggggttagc ggcacgcaat 60tgctatagtg agtcgtatta 80 11 39 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Primer 11 catggtggtt tgggttaggg ttagggttagggttaccac 39 12 18 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 12 taatacgact cactatag 18

What is claimed is:
 1. A method of reducing proliferative capacity of acell comprising contacting said cell with a compound or a salt thereofor a stereoisomer of compound I that has the formula:

where R¹ and R⁴ are independently -L-A where L is a linking group havingthe formula:

where n is 1-3; and each R5 is independently H, Me, OH, or OMe;

where R5 is as before and Y is O, S, SO, SO₂, NH, NMe, or NCOMe;

where R5 and Y are as before and X is CH₂, O, S, SO, SO₂, NH, NMe, orNCOMe;

where R⁶, R⁷, R⁸, and R⁹ are independently H, OMe, OEt, halogen, or Me;and A is a compound of the formula:

where m is 0-5 and R6 is halogen, NH₂, NO₂, CN, OMe, SO₂NH2, amidino,guanidino, or Me;

where o is 0-1; p is 0-2; q is 1-2 provided that when o+q is 2, in whichcase a pyrrolidine or pyrrole ring is indicated, or 3, in which apiperidine or pyridine ring is indicated; r is 0-3; R⁷ is H or Me; R⁸ isindependently Me, NO₂, OH, CH₂OH or halogen, and when r is 2-3, twoadjacent R8 substituents are —(CH═CH)₂— or —(CH2)₄— to form an annulatedsix-membered ring;

where R⁹ is independently H, Me, and when R9 is O; s is 0-1; Z is CH₂,O, NH, NMe, NEt, N(Me)₂, N(Et)₂, or NCO₂Et;

where Q is N, CH, NMe, or NEt; X is O, S, NH, NMe or NEt; R¹⁰ and R¹¹are independently H, Me, CH₂CO₂Et, R¹⁰ and R¹¹ taken together are—(CH═CH)₂— or —(CH₂)₄—;

where t is 1-4 4; u is 0-4, and R12 is independently Me, OH,

CO₂R¹³, CON(R¹³)₂, SO₃H, SO₂N(R¹³)₂, CN, CH(CO₂R¹³)₂, CH(CON(R¹³)₂)₂,N(R¹³)₂, or N(R¹³)₃ where R¹³ is H, Me, Et, or CH₂CH₂OH; and R², R^(2′),R^(2″), R^(″); R³, R^(3′), R^(3″), R^(3′″) are each independently H,OMe, halogen, or NO₂.
 2. The method of claim 1, wherein the cell is amammalian cell.
 3. The method of claim 1, wherein the cell is a humancell.
 4. The method of claim 1, wherein the cell is a cancer cell. 5.The method of claim 1, wherein said malignant cell is a breast cancercell, a prostate cancer cell, liver cancer cell, a pancreatic cancercell, a lung cancer cell, a brain cancer cell, an ovarian cancer cell, auterine cancer cell, a testicular cancer cell, a skin cancer cell, aleukemia cell, a head and neck cancer cell, an esophageal cancer cell, astomach cancer cell, a colon cancer cell, a retinal cancer cell, abladder cancer cell, an anal cancer cell and a rectal cancer cell.
 6. Amethod of reducing telomeric extension comprising administering acompound of claim 1 to a telomerase in the presence of a telomerasesubstrate.
 7. The method of claim 6, where the telomerase is in a cell.8. The method of claim 1, wherein said compound further promotesapoptosis.
 9. The method of claim 1, wherein said compound furtherpromotes apoptosis in a cell.
 10. The method of claim 1, wherein thecompound is a perylene compound.
 11. The method of claim 1, wherein thecompound is N,N′-bis(2-piperdinoethyl)-3,4,9,10-perylenetetracarboxylicacid diimide.
 12. The method of claim 1, wherein the compound isN,N′-bis(2-dimethylaminoethyl)-3,4,9,10-perylenetetracarboxylic aciddiimide.
 13. A compound of the formula

where R¹ and R⁴ are independently -L-A where L is a linking group havingthe formula:

where n is 1-3; and each R5 is independently H, Me, OH, or OMe;

where R5 is as before and Y is O, S, SO, SO₂, NH, NMe, or NCOMe;

where R5 and Y are as before and X is CH₂, O, S, SO, SO₂, NH, NMe, orNCOMe;

where R⁶, R⁷, R⁸, and R⁹ are independently H, OMe, OEt, halogen, or Me;and A is a compound of the formula:

where m is 0-5 and R⁶ is halogen, NH₂, NO₂, CN, OMe, SO₂NH₂, amidino,guanidino, or Me;

where o is 0-1; p is 0-2; q is 1-2 provided that when o+q is 2, in whichcase a pyrrolidine or pyrrole ring is indicated, or 3, in which apiperidine or pyridine ring is indicated; r is 0-3; R⁷ is H or Me; R⁸ isindependently Me, NO₂, OH, CH₂OH or halogen, and when r is 2-3, twoadjacent R⁸ substituents are —(CH═CH)2- or —(CH2)4- to form an annulatedsix-membered ring;

where R⁹ is independently H, Me, and when R9 is O; s is 0-1; Z is CH₂,O, NH, NMe, NEt, N(Me)₂, N(Et)₂, or NCO₂Et;

where Q is N, CH, NMe, or NEt; X is O, S, NH, NMe or NEt; R¹⁰ and R¹¹are independently H, Me, CH₂CO₂Et, R¹⁰ and R¹¹ taken together are—(CH═CH)₂— or —(CH₂)₄;

where t is 1-4 4; u is 0-4, and R¹² is independently Me, OH,

CO₂R¹³, CON(R¹³)₂, SO₃H, SO₂N(R¹³)₂, CN, CH(CO₂R¹³)₂, CH(CON(R¹³)₂)₂,N(R¹³)₂, or N(R¹³)₃ where R¹³ is H, Me, Et, or CH₂CH₂OH; and R², R^(2′),R^(2″), R^(2″); R³, R^(3′), R^(3″, R) ^(3′″) are each independently H,OMe, halogen, or NO₂.
 14. A method of reducing proliferative capacity ofa cell comprising contacting said cell with a compound having theformula II or a salt thereof or a stereoisomer of said compound:

where C is —CH═CH—, —(CH═CH)₂—, —(CH═CH)₃—, p-phenylene, o-phenylene,p-phenylene-CH═CH—, or o-phenylene-CH═CH—; B is O, S, or NR, and R is rMe or Et.
 15. The method of claim 14, wherein the cell is a mammaliancell.
 16. The method of claim 14, wherein the cell is a human cell. 17.The method of claim 14, wherein the cell is a cancer cell.
 18. Themethod of claim 14, wherein said cancer cell is a breast cancer cell, aprostate cancer cell, liver cancer cell, a pancreatic cancer cell, alung cancer cell, a brain cancer cell, an ovarian cancer cell, a uterinecancer cell, a testicular cancer cell, a skin cancer cell, a leukemiacell, a head and neck cancer cell, an esophageal cancer cell, a stomachcancer cell, a colon cancer cell, a retinal cancer cell, a bladdercancer cell, an anal cancer cell and a rectal cancer cell.
 19. A methodof reducing telomeric extension comprising administering a compound ofclaim 14, to a telomerase in the presence of a telomerase substrate. 20.The method of claim 19, where telomerase is in a cell.
 21. The method ofclaim 14, wherein said compound further promotes apoptosis in a cell.22. The method of claim 14, wherein the compound is a carbocyanine. 23.The method of claim 22, wherein the carbocyanine is3,3′-diethyloxadicarbocyanine (DODC).
 24. A method for identifying acandidate compound that inhibits telomerase activity, comprising thesteps: a) obtaining the three-dimensional structure of a selectedcompound; and b) determining the complementarity of the compound totelomere DNA G-quadruplex wherein a compound that exhibits at least 75%of the favourable intermolecular interaction energy of the perylenediimide 2-d(TTAGGG)₄ complex structure is indicated to inhibittelomerase activity.
 25. A method of identifying a telomerase inhibitorcomprising: a) contacting a compound with DNA G-quadruplex; and b)determining the melting point of the DNA G-quadruplex wherein a compoundexhibiting an increase in melting point of said quadruplex, relative tounbound DNA G-quadruplex, is indicated to inhibit telomerase activity.26. A method of identifying a telomerase inhibitor comprising the steps:a) preparing a DNA G-quadruplex/dye complex wherein the dye is boundwith the G-quadruplex; b) contacting said complex with a candidatecompound; and c) determining displacement of said dye in the complex bysaid candidate, wherein displacement of the dye identifies the candidateas a telomerase inhibitor.
 27. A method of identifying a telomeraseinhibitor comprising: a) contacting a candidate compound to beidentified as a telomerase inhibitor with DNA G-quadruplex; and b)determining the fluorescence or UV/VIS spectrum of the compound whereinan increase or decrease of the UV/VIS absorption or fluorescenceemission intensity of said compound relative to the UV/VIS absorption orfluorescence emission intensity in the absence of DNA-G-quadruplexindicates telomerase inhibitory activity of the compound.
 28. A compoundof the formula:

in which C is —CH═CH—, —(CH═CH)₂—, —(CH═CH)₃—, p-phenylene, o-phenylene,p-phenylene-CH═CH—, or o-phenylene-CH═CH—; B is O, S, or NR, and R is Meor Et.
 29. The method of claim 1, wherein the mitotic division of a cellis inhibited.
 30. The method of claim 14, wherein the mitotic divisionof a cell is inhibited.
 31. A compound of claim 28, having thestructure:


32. The method of claim 14, having the structure:


33. A compound of claim 13, having the formula:


34. The method of claim 1, having the formula:


35. The compound of claim 13, having the formula:


36. The method of claim 1, having the structure: